Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
  • A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
  • C07K16/241—Tumor Necrosis Factors
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/2863—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for growth factors, growth regulators
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/2887—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against CD20
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/32—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
  • A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/10—Immunoglobulins specific features characterized by their source of isolation or production
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
  • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance

Definitions

• the invention relates to sugars, in particular sucrose, their use in pharmaceutical compositions, and further to nanoparticulate impurities comprised in sugars.
• the invention further relates to methods for obtaining sucrose with reduced levels of the nanoparticulate impurities and related methods of preparing pharmaceutical compositions.
• NPI nanoparticulate impurities
• the signal is not caused by the dissolved sugar molecules whose size is only about 0.5 nm and 1 nm for mono- and disaccharides, respectively. Other authors had also noticed this interference signal in the past, but did not investigate or identify its cause or potential effects on pharmaceutical compositions.
• the intensity of the interference signal and thus the amount, or count of NPI particles differs between sugar types (e.g. trehalose, fructose maltose and galactose), sucrose of various purity grades, obtained from different suppliers, and different batches of the same supplier; the particle size distribution on the other hand does not vary much.
• sugar types e.g. trehalose, fructose maltose and galactose
• sucrose of various purity grades obtained from different suppliers, and different batches of the same supplier; the particle size distribution on the other hand does not vary much.
• WO01/36690A1 While being aimed at removing invert sugars from a sucrose-containing feed solution by nanofiltration, WO01/36690A1 is altogether silent on water-insoluble nanoparticulate impurities exhibiting a particle size of 20-500 nm and/or endotoxin-like properties.
• WO2015/191110A1 addresses the removal of endotoxins in the narrower sense (i.e., lipopolysaccharides) from aqueous sugar solutions using anion exchange chromatography in a polyethyleneimine (PEI) column, said endotoxins are neither explicitly nor implicitly described to be water-insoluble nanoparticles of any specific size.
• sugars may comprise a variety of impurities without any structural and/or compositional relationship to one another. This is problematic considering the use of sugars in pharmaceutical compositions.
• sugars and in particular sucrose and trehalose, are often employed in pharmaceutical compositions comprising polypeptide-based biologically active ingredients because they are preferentially excluded from the protein's surface, thereby increasing the free energy of the system and promoting conformational stability. Furthermore, sugars such as sucrose are used extensively as cryoprotectors and lyoprotectors for lyophilized protein compositions.
• the present invention is based on the inventors' discovery that NPI may present a significant threat to the performance and stability of pharmaceutical compositions comprising sucrose and without control of the NPI content there is a substantial risk of not being able to manufacture batches of such compositions which have consistent quality.
• the invention provides a method of preparing ultrapure sucrose, the method comprising the steps of (b) providing a quantity of a first sucrose comprising water-insoluble nanoparticulate impurities (NPI) at a level of 10 6 nanoparticles per gram of sucrose or higher, wherein the nanoparticles have a particle size ranging from 20 nm to 500 nm, comprise a (1-3)- ⁇ -glucan, and exhibit endotoxin-like properties; wherein prior to step (b), a step (a) is conducted of obtaining or determining the level of NPI in the first sucrose; and then (c) reducing the level of NPI to 10 5 nanoparticles per gram of sucrose or lower by (i) separating at least some of the NPI from the sucrose, optionally by filtration, centrifugation, crystallisation or chromatography of a solution of said sucrose; or by (ii) diluting the first sucrose with a second sucrose, the second sucrose
• the method may further comprise a step (d) of concentrating the sucrose solution of step (i) to a concentration of 10 % (w/v) or higher, or of drying the sucrose solution such as to obtain dry solid sucrose.
• the method may further comprise a step of obtaining or determining the level of NPI in the first sucrose, optionally by nanoparticle tracking analysis (NTA).
• NPI nanoparticle tracking analysis
• the water-insoluble NPI particles have a particle size ranging from 20 nm to 500 nm.
• the NPI may have a particle size distribution (PSD) characterised by a D50 value from 80 nm to 300 nm, in particular from 100 nm to 200 nm, as measured by nanoparticle tracking analysis (NTA) of an aqueous solution of the sucrose.
• PSD particle size distribution
• the water-insoluble NPI particles further exhibit endotoxin-like properties; i.e. they provide a positive test result in endotoxin tests such the Limulus Amebocyte Lysate tests (LAL) and/or modified LAL tests.
• the NPI comprise an immune-modulating (1-3)- ⁇ -glucan, as may be determined, for instance, by a modified LAL tests using reagents with eliminated Factor C, such as the (1-3)- ⁇ -glucan specific Glucatell ® test kit.
• the NPI may further comprise a dextran, optionally crosslinked and/or exhibiting a mean molecular mass from 10 kDa to 100 kDa.
• the NPI may comprise one or more organic colourants and/or an inorganic compound selected from silicium, aluminium, calcium, magnesium, phosphorus, sulphur, potassium, iron, or any combination of any of these.
• the method may be conducted such as to reduce the level of NPI to not more than 10 3 nanoparticles per gram of sucrose, or not more than 10 2 nanoparticles per gram of sucrose; or even such that the sucrose is substantially free of NPI, as can be determined by any suitable light-scattering based analytical techniques, such as nanoparticle tracking analysis (NTA) or electric zone sensing using a so-called Coulter counter.
• NTA nanoparticle tracking analysis
• Coulter counter so-called Coulter counter.
• the level of NPI is reduced via filtration or centrifugation, respectively, this may be performed by subjecting an aqueous solution of the sucrose to an ultrafiltration or ultradiafiltration process or to ultracentrifugation, respectively.
• ultrafiltration may be performed through a filter with a pore size of 0.02 ⁇ m or smaller.
• the invention in a second aspect, relates to an ultrapure sucrose as obtainable by the method described above.
• the invention further provides a solid, crystalline sucrose comprising water-insoluble nanoparticulate impurities (NPI) at a level of not more than 10 5 nanoparticles per gram of sucrose as determined by nanoparticle tracking analysis (NTA), preferably not more than 10 3 nanoparticles per gram of sucrose, and more preferably not more than 10 2 nanoparticles per gram of sucrose.
• NPI nanoparticulate impurities
• the invention relates to the use of such ultrapure sucrose for the preparation of a pharmaceutical or diagnostic composition
• a pharmaceutical or diagnostic composition comprising an active ingredient, preferably an active ingredient such as a peptide, a protein, a conjugate comprising a peptide or protein, a lipopeptide, or a lipoprotein; an active ingredient prone to aggregation and/or degradation; and/or an active ingredient sensitive to heat, mechanical stress, oxygen, acidic and/or basic conditions.
• the invention relates to a method of preparing a pharmaceutical or diagnostic composition comprising an active ingredient and sucrose, the method comprising the steps of obtaining or determining the level of NPI in the sucrose, optionally by nanoparticle tracking analysis (NTA); then, if the level of NPI in the sucrose is 10 6 nanoparticles per gram of sucrose or higher, or if the level of NPI in the sucrose is such that it would result in a level of NPI of 10 5 nanoparticles per gram of composition or higher, reducing the level of NPI to 10 5 nanoparticles per gram of sucrose or lower by (i) separating at least some of the NPI from the sucrose, optionally by filtration, centrifugation, crystallisation or chromatography; or by (ii) diluting the first sucrose with a second sucrose, the second sucrose comprising NPI at a level below 10 5 nanoparticles per gram of sucrose; and finally preparing the pharmaceutical or diagnostic composition incorporating the sucrose.
• NPI nano
• This method is particularly useful when preparing a plurality of consecutive batches of the pharmaceutical or diagnostic composition in the described manner, optionally using different batches of sucrose.
• the method may result in a higher reproducibility in terms of quality, purity and stability of the batches than currently achievable with sucrose-containing compositions.
• the pharmaceutical or diagnostic composition prepared with this method may, for instance, be in the form of a powder or a lyophilised solid foam for reconstitution, or in the form of an aqueous liquid.
• the invention relates to a pharmaceutical or diagnostic composition as obtained by the above described preparation method for use in the diagnosis or therapy of a human or a non-human animal.
• the pharmaceutical or diagnostic composition may comprise a peptide, a protein, a conjugate comprising a peptide or protein, a lipopeptide, or a lipoprotein as the active ingredient; and/or an active ingredient prone to aggregation and/or degradation; and/or an active ingredient sensitive to heat, mechanical stress, oxygen, acidic and/or basic conditions.
• the pharmaceutical or diagnostic composition may comprise a monoclonal antibody, and/or the sucrose in the pharmaceutical or diagnostic composition is substantially free of NPI, as determined by nanoparticle tracking analysis (NTA).
• nanoparticulate impurities means impurities which are in an aqueous environment, in particular at a physiologically compatible pH, such as within the range of pH 4 to pH 9 or from pH 5 to pH 8, detectable as nanoparticles having a particle size of 20 nm to 500 nm, e.g., as determined by laser diffraction or dynamic light scattering.
• the NPI nanoparticles are characterised by exhibiting an endotoxin-like immunoreactivity, which may be detected or determined, for example, with a Limulus test, also known as Limulus Amebocyte Lysate (LAL) test, or by any other test for identifying endotoxin-like properties, such as a modified LAL-test in which the Factor C is eliminated (e.g. Glucatell ® test).
• a Limulus test also known as Limulus Amebocyte Lysate (LAL) test
• LAL Limulus Amebocyte Lysate
• endotoxins are, according to a narrow definition, lipopolysaccharides found in the outer membrane of Gram-negative bacteria which trigger immune responses in animals, also other compounds which may not necessarily be derived from bacteria only (such as (1-3)- ⁇ -glucan which is also found in yeast or fungi) may exhibit immune-modulating properties and can be detected and quantified e.g. with the Glucatell ® test. Both bacterial endotoxins and (1-3)- ⁇ -glucan are also referred to as 'Pathogen-Associated Molecular Patterns' (PAMPs) in the literature.
• PAMPs 'Pathogen-Associated Molecular Patterns'
• water-insoluble means that the substance or substances forming the NPI nanoparticles are substantially less water-soluble than the sucrose, for which reason they are detectable as nanoparticles in an aqueous environment.
• ultrapure with respect to sucrose means that the sucrose exhibits a high degree of purity, also with respect to the level of NPI.
• the expression may also be understood as meaning that the sucrose does not incorporate NPI at a level which would detrimentally affect the quality, performance or stability of a pharmaceutical formulation comprising the sucrose.
• Sucrose comprising NPI at a level of not more than about 10 6 nanoparticles per g may also be considered as an ultrapure sucrose.
• Nanoparticulate refers to any type, form, structure or morphology of nanoparticle that may exist, in particular in an aqueous environment.
• the present invention provides a method of preparing ultrapure sucrose, the method comprising the steps of:
• the inventors have discovered that the NPI present in sucrose, even at a level at which the sucrose complies with all specifications provided by the major pharmacopoeias, may negatively affect the performance and stability of pharmaceutical compositions with polypeptide-based active ingredients. Moreover, the inventors found that in particular the batch-to-batch consistency of the performance and stability of such compositions may be adversely affected by variable NPI levels of sucrose. In the absence of such insights, it was entirely unexpected that by controlling the NPI content of the sucrose, e.g. by selecting a sucrose that has a low NPI content or by purifying sucrose with respect to the NPI content prior to its incorporation within a pharmaceutical composition, the product quality of the pharmaceutical compositions and its batch-to-batch consistency could be significantly increased.
• step (a) of obtaining or determining the level of NPI in the first sucrose which is conducted prior to step (b) is conducted by nanoparticle tracking analysis (NTA), as detailed for instance in the examples section below; however, any other particle size determination method which is capable of sensing and quantifying particles in the size range of 20 nm up 500 nm may be used as well, e.g. other light-scattering based analytical techniques like dynamic light scattering (DLS).
• NTA nanoparticle tracking analysis
• the NPI particles have a particle size distribution in an aqueous solution characterised by a D50 value from 80 nm to 300 nm as measured by nanoparticle tracking analysis (NTA), in particular from 100 nm to 200 nm, or from 125 nm to 165 nm.
• NTA nanoparticle tracking analysis
• the D90 value is from about 100 nm to about 400 nm as measured by nanoparticle tracking analysis (NTA), in particular from about 150 nm to about 350 nm, or from about 220 nm to about 290 nm.
• the D10 value is from about 50 nm to about 150 nm as measured by nanoparticle tracking analysis (NTA), in particular from about 70 nm to about 130 nm, or from about 80 nm to about 110 nm.
• the particle size distribution of the NPI particles is monomodal.
• the NPI comprise a (1-3)- ⁇ -glucan.
• This type of compound - which may be derived from the cell walls of bacteria, yeast and fungi - may contribute, or even be primarily responsible for the immunomodulatory or endotoxin-like properties of the NPI. This is one of the reasons why the level of NPI in sucrose, but also in pharmaceutical compositions comprising the sucrose, should be kept as low as possible.
• the presence and content of (1-3)- ⁇ -glucan in the NPI can be determined, for example, by a modified Limulus Amebocyte Lysate (LAL) test using a reagent with the Factor C being eliminated which renders this test more specific for (1-3)- ⁇ -glucan.
• LAL modified Limulus Amebocyte Lysate
• the (1-3)- ⁇ -glucans found in the NPI stems at least partially from the cell walls of microorganisms, e.g. Leuconostoc bacteria, which enter the sugar cane or beet e.g. during harvesting, cutting and grinding but also during later processing steps; however they may also stem from other sources such as fungi or yeast.
• microorganisms e.g. Leuconostoc bacteria, which enter the sugar cane or beet e.g. during harvesting, cutting and grinding but also during later processing steps; however they may also stem from other sources such as fungi or yeast.
• the method of the invention is particularly advantageous and leads to unexpected improvements in product quality, performance and stability for any pharmaceutical composition for whose manufacture the incorporation of a sucrose comprising such NPI is contemplated.
• the NPI comprise a dextran, wherein the dextran is optionally crosslinked and/or exhibits a mean molecular mass from 10 kDa to 100 kDa.
• the NPI comprise a (1-3)- ⁇ -glucan and a dextran, wherein the dextran is optionally crosslinked and/or exhibits a mean molecular mass from 10 kDa to 100 kDa
• the NPI may comprise one or more organic colourants and/or an inorganic compound selected from silicium, aluminium, calcium, magnesium, phosphorus, sulphur, potassium, iron, or any combination of any of these.
• the colourants are aromatic colourants, as may be detected or determined, for instance, by measuring the internal fluorescence of a sample.
• the colourants are aromatic colourants exhibit two patterns of maximum fluorescence intensity; pattern 1 at about 280/390 nm (which may potentially be caused by two fluorophores with a close similarity to tryptophan and tyrosine) and pattern 2 at about 340/420 nm (which may potentially be caused by catechols formed by base-catalysed sugar degradation).
• step (c) is conducted such as to reduce the level of NPI to not more than 10 3 nanoparticles per gram of sucrose, or not more than 10 2 nanoparticles per gram of sucrose; or such that the sucrose is substantially free of NPI, as determined by light-scattering based analytical techniques, such as dynamic light scattering (DLS) or nanoparticle tracking analysis (NTA).
• DLS dynamic light scattering
• NTA nanoparticle tracking analysis
• step (c) may be performed such as to reduce the level of NPI particles per g of sucrose by the factor of 100 or higher.
• step (c) is performed such as to reduce the NPI particles by the factor of at least 200, or at least 300, or at least 500, or at least 1,000, or at least 2,000, or at least 3,000, or at least 5,000, or at least 10,000, respectively.
• the filtration in step (c)(i) is performed as an ultrafiltration or ultradiafiltration of an aqueous solution of the first sucrose.
• the aqueous solution of the first sucrose may be subjected to ultrafiltration through a filter with a pore size of approx. 0.02 ⁇ m or smaller, such as a PVDF filter with a nominal pore size down to 0.001 ⁇ m; or through a membrane with a cut-off of 30 kDa or smaller. Filtration through a 0.02 ⁇ m filter decreases the NPI related signal in both DLS and NTA measurements to background levels, i.e. not showing a peak at about 100-200 nm anymore.
• Filters with larger pore sizes such as common sterile filtration filters (often 0.2 ⁇ m or 0.22 ⁇ m) or even 0.1 ⁇ m filters were found to be substantially less efficient in reducing the level of NPI in aqueous sucrose solutions.
• the aqueous solution of the first sucrose may be subjected to ultradiafiltration in a tangential flow filtration (TFF) system equipped with a TFF-membrane; for instance, a TFF-membrane with a cut-off of 30 kDa or smaller.
• TFF tangential flow filtration
• Such ultradiafiltration systems have been used by the inventors in order to obtain and/or purify NPI from aqueous sucrose solutions, e.g. in order to investigate the size and composition of NPI and /or their level per gram sucrose.
• the centrifugation in step (c)(i) is performed as ultracentrifugation of an aqueous solution of the first sucrose.
• centrifugal filter units may be employed during ultracentrifugation, e.g. units equipped with a filter membrane having a cut-off of 30 kDa or 10 kDa. Such centrifugal filter units have also been used by the inventors in order to obtain and/or purify NPI from aqueous sucrose solutions, in particular for concentrating NPI-containing samples after ultradiafiltration.
• a sucrose of acceptable NPI level may also be obtained by diluting the first sucrose with a second sucrose, the second sucrose comprising NPI at a level below 10 5 nanoparticles per gram of sucrose; or in other words by mixing a 'high-NPI sucrose' with a 'low-NPI sucrose' until the total content of the mixture falls below 10 5 nanoparticles per gram of sucrose.
• this dilution step is advisable and economically reasonable only in cases where the second sucrose exhibits an NPI-content sufficiently low to 'counter-act' the higher NPI-content in the first sucrose.
• the first and second sucrose comprise 10 6 and 10 1 nanoparticles per gram of sucrose, respectively, a 1:10 mixture would suffice to reduce the NPI in the mixture to an acceptable level of below 10 5 nanoparticles per gram of sucrose.
• This reasonable dilution or mixing ratio would allow manufacturers of pharmaceutical grade sugars - within given boundaries - to account for inherent differences in their raw materials which may have an effect on the final NPI level.
• the dextran content in the raw material is typically guided by the load of Leuconostoc bacteria as described earlier.
• the first sucrose comprises a higher number of NPI, such as 10 9 nanoparticles per gram of sucrose already (a content as found in pharmaceutical grade sucrose by the inventors in an earlier study)
• a substantially NPI-free second sucrose would require very large amounts of said second sucrose; in which case it may be economically more reasonable to remove or reduce these NPI from the first sucrose by one of the other steps, such as filtration, centrifugation, crystallisation or chromatography, rather than attempting a dilution.
• the invention also provides the sucrose as obtainable by the method(s) described above; i.e. a purified sucrose from which the NPI were substantially removed or in which the level of NPI has been reduced to 10 5 nanoparticles per gram of sucrose or lower.
• a purified sucrose from which the NPI were substantially removed or in which the level of NPI has been reduced to 10 5 nanoparticles per gram of sucrose or lower Such ultrapure sucrose offers a number of advantages, in particular when incorporated into pharmaceutical or diagnostic compositions. For instance, ultrapure sucrose introduces less NPI and in consequence less (1-3)- ⁇ -glucan (as comprised in the NPI) and less nanoparticles which could potentially act as an initial nucleus, or nucleation site, for crystallisation and/or aggregation processes, especially in aqueous compositions.
• the invention also provides ultrapure, solid, crystalline sucrose comprising water-insoluble nanoparticulate impurities (NPI) at a level of not more than 10 5 nanoparticles per gram of sucrose as determined by nanoparticle tracking analysis (NTA).
• NPI nanoparticle tracking analysis
• the ultrapure, solid, crystalline sucrose comprises not more than about 10 4 nanoparticles per gram of sucrose, not more than about 10 3 nanoparticles per gram of sucrose; or not more than about 10 2 nanoparticles per gram of sucrose, respectively.
• the invention provides the use of ultrapure sucrose (i.e. sucrose comprising not more than 10 5 nanoparticles per gram of sucrose) for the preparation of a pharmaceutical or diagnostic composition comprising an active ingredient, or drug.
• ultrapure sucrose i.e. sucrose comprising not more than 10 5 nanoparticles per gram of sucrose
• the use of ultrapure sucrose offers number of benefits when incorporated in such compositions.
• the ultrapure sucrose comprises not more than about 10 4 nanoparticles per gram of sucrose, not more than about 10 3 nanoparticles per gram of sucrose; or not more than about 10 2 nanoparticles per gram of sucrose, respectively.
• the active ingredient in these pharmaceutical or diagnostic compositions is (a) a peptide, a protein, a conjugate comprising a peptide or protein, a lipopeptide, or a lipoprotein; (b) prone to aggregation and/or degradation; and/or (c) sensitive to heat, mechanical stress, oxygen, acidic and/or basic conditions.
• Proteins have an inherent propensity to aggregate (and ultimately precipitate) because their structure is dynamic and mainly held together by a combination of van der Waals forces, disulfide linkages, hydrogen bonds, etc., and as aggregation proceeds, the activity of the protein molecules may diminish or be lost, thereby reducing the efficacy of the composition.
• compositions comprising proteins and/or other active ingredients prone to aggregation and/or degradation may benefit in particular from the use of ultrapure sucrose.
• sucrose itself may be considered as the active ingredient, or at last as one of the active ingredients; for instance, where the pharmaceutical composition is an oral solution (such as a 24 % sucrose oral solution for pain relief in neonates) or a composition for parenteral nutrition.
• the pharmaceutical composition is an oral solution (such as a 24 % sucrose oral solution for pain relief in neonates) or a composition for parenteral nutrition.
• the latter may benefit from the use of ultrapure sucrose, since the (1-3)- ⁇ -glucan related immunogenicity is typically more pronounced with parenteral than with oral compositions.
• the invention therefore provides a method of preparing a pharmaceutical or diagnostic composition comprising an active ingredient and sucrose, the method comprising the steps of
• the pharmaceutical or diagnostic composition prepared by the above method is provided in the form of (a) a powder or a lyophilised solid foam for reconstitution, or (b) an aqueous liquid.
• a powder or a lyophilised solid foam for reconstitution or (b) an aqueous liquid.
• the provided level of NPI refers to the final composition; i.e. in the case of a powder or a lyophilised solid foam for reconstitution it is intended to refer to the reconstituted composition.
• sucrose content in common, currently marketed products for reconstitution such as Enbrel ® , Serostim ® , Remicade ® or Stelara ® is typically below 10 % in the reconstituted solutions, ranging from about 1 % to about 8 %, such that it is advisable to reduce the level of NPI in the composition below 10 5 nanoparticles per gram of composition, preferably below 10 4 nanoparticles per gram of composition and more preferably below 10 3 nanoparticles per gram of composition.
• the level of NPI will be reduced to 10 3 nanoparticles per gram of sucrose or lower using the above mentioned steps (i) or (ii) if the level of NPI in the sucrose is such that it would result in a level of NPI of 10 5 nanoparticles per gram of composition or higher.
• the separation of the NPI from the sucrose in step (i) of the above preparation method by filtration, centrifugation, crystallisation or chromatography is performed such as to reduce the number of nanoparticles below the detection limit of the respective method, e.g. NTA; i.e. such that the sucrose is substantially free of NPI when incorporating it into the pharmaceutical or diagnostic composition along with the active ingredient.
• NPI levels chosen herein in terms of maximum levels accepted per unit of sucrose or per unit of a composition are predominantly provided as guidance and may be adapted to specific situations or products by the skilled person. It will be easily understood that a composition which contains only rather low amounts of sucrose, such as about 2 % or lower, may tolerate a higher NPI level than a composition with rather high sucrose contents, such as about 10 % or higher. Similarly, a composition comprising aggregation sensitive compounds such as proteins or comprising supersaturated drugs prone to precipitation may be more sensitive to the NPI level of the sucrose used than other compositions in which the NPI are less likely to trigger aggregation or crystallisation. Also, a paediatric composition intended for neonates or infants may require smaller NPI levels than compositions for adults, since infants will typically react more sensitive to endotoxins such as the (1-3)- ⁇ -glucan comprised in the NPI.
• a plurality of consecutive batches of the pharmaceutical or diagnostic composition is prepared, and each of these batches is prepared following the steps of the above described method.
• different batches of sucrose may be used.
• Preparing a plurality of consecutive batches is very common in the field of preparing pharmaceutic or diagnostic compositions. It is also common that different batches, or lots, of the required raw materials are used. As the inventors have shown, the level of NPI found in sucrose varies. Hence, removing or reducing the level of NPI to 10 5 nanoparticles per gram of sucrose or even lower helps to ensure and/or improve the batch-to-batch reproducibility of the pharmaceutical or diagnostic compositions prepared.
• the plurality of consecutive batches will exhibit less variability in terms of quality, purity and stability parameters, such as content and activity of the drug or number of aggregates formed at time to (after preparation) and after times t 1 to t n during storage under ambient and/or accelerated conditions.
• the invention therefore provides a pharmaceutical or diagnostic composition as obtained by the above preparation method for use in the diagnosis or therapy of a human or a non-human animal.
• the active ingredient used in the diagnosis or therapy is (a) a peptide, a protein, a conjugate comprising a peptide or protein, a lipopeptide, or a lipoprotein; (b) an active ingredient prone to aggregation and/or degradation; and/or (c) an active ingredient sensitive to heat, mechanical stress, oxygen, acidic and/or basic conditions.
• the active ingredient in the pharmaceutical or diagnostic composition is a monoclonal antibody; and/or the sucrose pharmaceutical or diagnostic composition is substantially free of NPI, as determined by nanoparticle tracking analysis (NTA).
• NPI nanoparticle tracking analysis
• Herceptin ® (trastuzumab), MabThera ® (rituximab), Remicade ® (infliximab), and Erbitux ® (cetuximab) were donated by local hospitals.
• Pharmaceutical-grade sucrose (Ph.Eur.) was purchased from VWR BDH Prolabo ® (Bruchsal, Germany) and Südzucker (Mannheim, Germany).
• Hisitidine-HCl, trehalose, sodium citrate, sodium chloride, sodium dihydrogen phosphate, di-sodium hydrogen phosphate, and citric acid were purchased from Merck (Darmstadt, Germany).
• Polysorbate 20 and 80, and glycine was purchased from Sigma (Taufmün, Germany).
• Histidine was purchased from Amresco (Solon, Ohio, USA).
• PVDF syringe and membrane filters with a pore size of 0.2 ⁇ m were obtained from Millipore (Schwalbach, Germany).
• NPI particles were obtained from pharmaceutical-grade sucrose batches by diafiltration of 1 L freshly prepared aqueous sucrose solution 50 % (w/v) using a Minimate II Tangential Flow Filtration (TFF) system equipped with a 30 kDa TFF membrane (both from Pall, Crailsheim, Germany).
• TFF Tangential Flow Filtration
• the retentate was concentrated to about 300 mL and subsequently diafiltrated with MilliQ ® -water until a diafiltration volume DV of 14 was achieved (ratio between filtrate volume and retentate volume).
• the retentate was concentrated to about 50 mL and filtered with a syringe filter (0.2 ⁇ m) before the retentate was further concentrated to about 1 mL with Amicon Ultra 15 centrifugal filter-units (Millipore) with a molecular cut-off of 10 kDa.
• the NPI sample was aliquoted into 0.5 mL cryotubes and finally stored at -80 °C until further use.
• the final sample contained 7 ⁇ 10 11 nanoparticles/mL, the particles exhibiting a size range of about 100-200 nm and a monomodal PSD, as determined by NTA (see below).
• NPI nanoparticle tracking analysis
• a NanoSight LM20 NanoSight, Amesbury, UK
• a 405 nm blue laser 405 nm blue laser
• a sample chamber 405 nm blue laser
• a Viton fluoroelastomer O-ring 405 nm blue laser
• samples are loaded into the sample chamber and subsequently analysed in triplicate at stopped flow. Between each repetition, 0.1 mL of sample is flushed through the chamber.
• the NTA 2.3 software is used for capturing and analysing the data; and movements of the particles in the samples are recorded as videos for 60 s, while the shutter and gain settings of the camera are set automatically by the software for an optimal particle resolution. If sample dilution is necessary to achieve a more optimised concentration for NTA, ultrapure water of Type 1 as defined in e.g. ISO 3696 (such as Milli-Q ® water or MQ-water) is used as a diluent and all results calculated back to the original concentration.
• ultrapure water of Type 1 as defined in e.g. ISO 3696 such as Milli-Q ® water or MQ-water
• the zeta-potential of the NPI particles was measured by laser Doppler electrophoresis (LDE) by using a Zetasizer Nano ZS (Malvern Instruments, Worcestershire, UK) and the Zetasizer software version 7.03 for system control and data acquisition.
• NPI samples at a concentration of 5 ⁇ 10 11 nanoparticles /mL (based on NTA) were buffered at pH 7.4 (1 mM phosphate buffer) or at pH 3.0 (1 mM citrate buffer) and 1000 ⁇ L sample transferred into a folded capillary cell (Malvern) for measurement.
• Each zeta-potential measurement is the average of three measurements consisting of 100 sub runs.
• the LDE evaluation reconfirmed earlier results in that the NPI particles exhibited a zeta potential of -13.4 ⁇ 1.6 (at pH 7.4) and -8.0 ⁇ 0.3 mV (at pH 3.0).
• the (1-3)- ⁇ -glucan level in the NPI particles is measured using a Glucatell ® kit (Cape Cod, East Falmouth, USA), according to the instructions of the manufacturer.
• the NPI containing samples are diluted with endotoxin free water in four 10-fold serial dilution steps under aseptic and particle free conditions.
• the mixture of the (diluted) sample and the Glucatell ® -reagent is placed in a microplate-heating block at 37 °C for the recommended time.
• a volume of 50 ⁇ L from each of the three diazo reagents is added to the mixture to stop the reaction.
• the absorbance at 545 nm is measured and (1-3)- ⁇ -glucan concentration in pg/mL is calculated based on a standard curve.
• the NPI particles gave a strong signal for (1-3)- ⁇ -glucan, namely 750 ng/mL for the NPI sample with about 7 ⁇ 10 11 nanoparticles/mL, compared to only 0.013 ng/mL for the control sample.
• trastuzumab, rituximab, infliximab, and cetuximab were diluted to a monoclonal antibody (mAbs) concentration of 2 mg/mL using the following formulation buffers (all filtered through 0.2 ⁇ m PVDF syringe filters):
• samples of all four NPI levels were stored at 40 °C for one week for a concentration-dependency study. Sample handling was performed under laminar airflow conditions.
• the vials were independently tested by two trained examiners for presence or absence of visible particles or turbidity under gentle, manual, radial agitation for 5 s in front of a white background and for 5 s in front of a black background.
• MFI Micro-flow imaging
• an MFI5200 system ProteinSimple, Santa Clara, CA, USA
• MVSS MFI View System Software
• the system was flushed with 10 mL purified water at maximum flow rate and flow cell cleanliness was checked visually between measurements.
• the respective formulation buffer was used as a blank to perform illumination optimization prior to each measurement. Samples of 0.5 mL with a pre-run volume of 0.2 mL were analysed at a flow rate of 0.17 mL/min and a fixed camera rate (not adjustable by the user), leading to a sampling efficiency of about 80-85 %.
• Size-exclusion chromatography was performed on a TSK Gel 4000 SWXL column (300 mm ⁇ 7.8 mm) (Tosoh Bioscience, Montgomeryville, USA) and an Agilent 1200 high-performance liquid chromatography system (Agilent Technologies, Palo Alto, USA) coupled to an ultraviolet (UV) detector set at 280 nm.
• the mobile phase was composed of 100 mM sodium phosphate, 100 mM sodium sulfate at a pH of 7.0.
• the flow rate was set to 0.6 mL/min. Samples were centrifuged at 10,000 g for 3 min, kept at 2-8 °C and injected in duplicates of each 25 ⁇ L.
• the sample recovery was calculated as the total peak area relative to the control-spiked sample at to. Peaks with a retention time above 20 min were buffer related and not considered. The monomer peak retention time was at 17.5 min. Peaks with a shorter retention time than the monomer were regarded as high molecular weight (HMW) species. In contrast, peaks with a longer retention time than the monomer were regarded as low molecular weight (LMW) species. Contents of monomer, HMW- and LMW species are given as percentages relative to the total sample recovery.
• Table 1 summarizes and ranks the observed effects of the NPIs on the stability of the tested antibodies. A higher score (sum of +'s) correlates to a more pronounced mAb degradation compared to the other tested mAbs.
• Rituximab was affected to the lowest extent by the presence of the NPIs, showing immediate formation of high numbers of nm-sized particles as the main degradation product. Cetuximab similarly showed immediate formation of high numbers of nm-sized particles as the main degradation product, but was overall degraded more severely than rituximab. Trastuzumab showed formation of high numbers of ⁇ m-sized particles as the main degradation product and the appearance of turbidity. Among the tested mAbs, infliximab was degraded to the greatest extent, showing high numbers of nm- and ⁇ m-sized particles as the main degradation product as well as the appearance of turbidity.
• trastuzumab and infliximab were affected directly after spiking NPI.
• degradation of rituximab and cetuximab was observed only after 14 weeks at elevated temperatures.
• stability of rituximab and infliximab was affected by NPI concentrations that are potentially present in final drug products.
• the stability of trastuzumab was only affected at elevated NPI concentrations.
• formulation buffer controls did not show instabilities in any of the tested parameters and the pH values measured immediately after sample preparation were within 0.1 pH units from those of the corresponding control samples. It can thus be excluded that the observations are purely due to differences in formulation buffer composition or a result of excipient instabilities and/or pH shifts.
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